Molecular imprinted colored silica beads

ABSTRACT

Macromolecular imprinted silica particles (“MIP”) in the presence of polymer grafted carbon black are disclosed. The disclosed molecular imprinted beads can detect disease in body fluids. For the silica gel matrix, tetraethyl orthosilicate (TEOS) was used as the backbone monomer and 3-aminopropy/triethoxysilane (APS) as a functional monomer. Carbon black was added to the sol-gel process, yielding black silica particles. Furthermore, sodium dodecyl sulfate (SDS) was used as a structure-directing agent to increase network diffusion of the template. A total of 16 MIPs were synthetized in parallel with variables that evaluate the role of key reactants in the synthesis procedure. Agglomeration tests were performed with all 16 MIPs in the presence of their template, alongside their respective controls using only phosphate buffered saline (“PBS”). Each of the MIPs was evaluated using a novel device capable of simultaneously measuring up to four samples for near infrared transmission.

CROSS-REFERENCE TO PROVISIONAL APPLICATION

This non-provisional patent application claims the benefit under 35U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No.61/862,719 filed on Aug. 6, 2013, entitled “MOLECULAR IMPRINTED COLOREDSILICA BEADS,” which is hereby incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The disclosed embodiments relate to imprinting polymers. The disclosedembodiments relate to molecular imprinting of high molecular weightcompounds. The disclosed embodiments also relate to colored silica beadswith imprinted antibody binding sites.

BACKGROUND

Molecular imprinting creates artificial receptors by the formation of apolymer network around a template molecule. Protein imprinting relatesto obtaining an imprint of a protein in a polymer network. Afterimprinting, imprinted proteins are washed away, degraded, or digested,thus leaving behind a cavity which preferentially binds that protein. Inpreviously proposed solutions, it has been difficult to produceimprinted mnicroparticles from the type of polymers used. Uncoloredbeads used in protein imprinting are difficult to use in antibodybinding assays.

Protein imprinting can be effective for molecules with low molecularweight (<1500 Da). Imprinting high molecular weight proteins orhormones, and compounds such as DNA, viruses, and bacteria withinpolymer matrices has been extremely challenging. For large templatemolecules, polymer crosslink densities seriously hinder mass transfer ofthe template, leading to slow template removal and rebinding kineticsor, in the worst case, permanent entrapment of the template in thepolymer network due to physical immobilization. In previous proposedsolutions, template rebinding is unreliably quantified, results are notevaluated critically, and often lack statistical analysis.Physicochemical properties such as charge or hydrophobicity can stronglyvary in dfferent regions of the protein template, whereas similarregions may be present in other templates. This could lead to aspecificbinding and cross-reactivity of the imprinted polymer. Furthermore, thesynthesis environment is usually too aggressive for the template, wherethe template solvent often denatures the template before an imprint isformed.

Therefore, a need exists for quantifiable imprinted molecules having ahigh molecular weight. The disclosed beads are utilized as diagnosticsin low resource settings as no temperature control is required.

SUMMARY

The following summary is provided to facilitate an understanding of someof the innovative features unique to the embodiments disclosed and isnot intended to be a full description. A full appreciation of thevarious aspects of the embodiments can be gained by taking the entirespecification, claims, drawings, and abstract as a whole.

The disclosed embodiments relate to imprinting polymers.

The disclosed embodiments relate to molecular imprinting of highmolecular weight compounds.

The disclosed embodiments further relate to colored silica beads withimprinted antibody binding sites.

The above and other aspects can be achieved as is now described. Acomposition of a macromolecular imprinted silica particles (“MIP”) inthe presence of polymer grafted carbon black is disclosed. The disclosedmolecular imprinted beads can detect disease in body fluids. For thesilica gel matrix, tetraethyl orthosilicate (“TEOS”) was used as thebackbone monomer and 3aminopropy/triethoxysilane (“APS”) as a functionalmonomer. Carbon black was added to the sol-gel process, yielding blacksilica particles. Furthermore, sodium dodecyl sulfate (“SOS”) was usedas a structure-directing agent to increase network diffusion of thetemplate. A total of 16 MIP's were synthetized in parallel withvariables that evaluate the role of key reactants in the synthesisprocedure. Agglomeration tests were performed with all 16 MIP's in thepresence of their template, alongside their respective controls usingonly phosphate buffered saline (“PBS”). Each of the MIPs was evaluatedusing a novel device capable of simultaneously measuring up to foursamples for near infrared transmission.

Agglomeration tests were performed using carbon black, UV, fluorescentred, and silica particles synthesized without carbon black in thepresence of their template. These tests were carried in parallelalongside various controls. Only carbon black silica particlesagglomerated in the presence of theft template, whereas UV red andparticles without carbon black settled independently regardless ofpresence of their template. The presence of the polymer-grafted carbonblack enables the synthesis of imprinted materials that can rebind highmolecular weight templates.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying figures, in which like reference numerals refer toidentical or functionally-similar elements throughout the separate viewsand which are incorporated in and form a part of the specification,further illustrate the embodiments and, together with the detaileddescription, serve to explain the embodiments disclosed herein.

FIG. 1 illustrates an exemplary pictorial illustration of creatingartificial receptors by the formation of a polymer network around atemplate molecule, according to a preferred embodiment;

FIG. 2 illustrates an exemplary pictorial illustration of hCG incubatedbeads binding to the hCG antibody in a nitrocellulose membrane, inaccordance with the disclosed embodiments;

FIG. 3 illustrates an exemplary graphical illustration of the near IRtransmission through a cuvette in the presence of dispersed beads foreach pair as function of time. In accordance with the disclosedembodiments;

FIG. 4A illustrates an exemplary pictorial illustration of SEM imagesdemonstrating surface morphology, specifically black anti-WN particlesat 1200×, showing microparticle shape, in accordance with the disclosedembodiments;

FIG. 4B illustrates an exemplary pictorial illustration of SEM imagesdemonstrating surface morphology, specifically black anti-WN particlesat 22,000× illustrating aggregate morphology of macroparticles, inaccordance with the disclosed embodiments;

FIG. 5 illustrates an exemplary pictorial illustration of agglomerationtest of UV red, carbon black, and non-colored silica particles, inaccordance with the disclosed embodiments; and

FIG. 6 illustrates an exemplary pictorial illustration of conical tubescontaining imprinted silica particles that were re-suspended afterovernight precipitation, in accordance with the disclosed embodiments.

DETAILED DESCRIPTION

The particular values and configurations discussed in these non-limitingexamples can be varied and are cited merely to illustrate at least oneembodiment and are not intended to limit the scope thereof.

The embodiments will now be described more fully hereinafter withreference to the accompanying drawings, in which illustrativeembodiments of the invention are shown. The embodiments disclosed hereincan be embodied in many different forms and should not be construed aslimited to the embodiments set forth herein; rather, these embodimentsare provided so that this disclosure will be thorough and complete, andwill fully convey the scope of the invention to those skilled in theart. Like numbers refer to like elements throughout. As used herein, theterm “and/or” includes any and all combinations of one or more of theassociated listed items.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an”, and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the relevant art andwill not be interpreted in an idealized or overly formal sense unlessexpressly so defined herein.

As illustrated in the exemplary pictorial illustration 100 of FIG. 1,molecular imprinting is a technique used to create artificial receptorsfor molecules or proteins by the formation of a polymer network around atemplate molecule. As shown in (A) of FIG. 1, the template, functionalmonomers, and crosslinker (+) form a pre-polymerization complex. In (B),polymerization of monomers and crosslinker fixes the complex. Finally,in (C), removal of the template leaves rebinding cavities.

To achieve macromolecular imprinting, the synthesis of macromolecularimprinted polymers (“MIPs”) must occur at an environment where thetemplate can maintain proper folding and 3D structures for sufficienttime frames to allow the formation of the imprints. Finally, the processmust allow recapturing the template for its reutilization. If newtemplate must be used for each synthesis, the key advantages of MIPssuch as scalability and cost effectiveness are eliminated; the processwould be more expensive than using traditional antibodies. There arecases, however, where the availability of MIPs available as sensors ormarkers trump the use of antibodies, for example, in remote and lowresourced settings or for defense applications.

The majority of imprinting technologies involve the synthesis andtesting of MIPs in organic solvents. This is necessary as to increasehydrogen bonding interactions between template and the MIR In aqueoussolutions, hydrogen bonding is reduced as well as the binding strengthof non-covalent template-monomer interactions in the imprinting. A MIPthat is capable of template recognition at aqueous environment is ofgreat importance as this greatly reduces materials and skills necessaryfor the recognition and separation of molecules of interest.

After selecting a target template for molecular imprinting, the nextstep is the selection of an appropriate polymer matrix, in which highaffinity binding sites can be created, ideally without introducingaspecific interactions. In the pre-polymer mixture, several possibleinteractions such as hydrophobic interactions, hydrogen bonds, Van derWaals forces, and electrostatic interactions determine the spatialarrangement of monomers around the protein template. This spatialarrangement is then fixed by polymerization of monomers and crosslinker.Biological macromolecules are very complex and possess many potentialrecognition sites at their surface, such as charged amino acids andhydrophobic hydrophilic regions. This makes the creation of molecularimprinted polymers with high selectivity challenging, due to possiblecross-reactivity with proteins with similar charge orhydrophobic/hydrophilic structure as the imprinted template protein.Removal of the template leaves a chemically and sterically complementaryvoid, or imprint, in the polymer network, which is able to rebind thetemplate.

An important step in the process of creating imprints with highselectivity and absorption capacity is the removal of the imprintedtemplate, especially because the imprint cavities of interest, or thosecavities with the highest binding affinity, will most strongly retainthe template molecules during template extraction. Moreover, diffusionand removal of high molecular weight proteins from imprinted polymers ischallenging.

In the disclosed embodiments, imprints of proteins in silica microbeadsare obtained from a mixture of 1.32 ml TEOS, 0.235 ml DI water, 0.33 mlof 0.1 M, and 0.4 ml of absolute ethanol, 0.33 ml of3-aminopropyltriethoxysilane (APS) mixed with 1.5 mg of Human chorionicgonadotropin (hCG), 0.5 ml of 0.1M SDS and 0.5 ml HP black ink. The twomixtures are combined in 10 ml of water, which causes the polymerizationto proceed immediately. The resulting polymers are washed, dried,ground, and separated by centrifugation. The resulting beads (1-5microns in diameter) are washed in acetic acid/methanol (50/50) 10-20times or until absorbance at 280 nm is below 0.04. The imprinted beadsare able to rebind hCG in an antibody-based pregnancy test, such as, forexample, First Response™ or EPT™.

FIG. 2 illustrates an exemplary pictorial illustration 200 of hCGincubated beads binding to the hCG antibody in a nitrocellulosemembrane, in accordance with the disclosed embodiments. This is asandwich assay, where ½ of the sandwich is the traditional antibodyassay. Antibodies are more expensive than the silica beads and expire athigh or low temperatures, whereas silica beads are very stable. Onecould also make magnetic, colored, imprinted beads using iron oxide inthe formulation. These beads may be used in a flow cytometer device, asdisclosed in U.S. patent application Ser. No. 13/849,124, which isincorporated by reference herein in its entirety.

Molecular imprinting utilizing sol-gel silica process in the presence ofpolymer grafted carbon black is disclosed. The presence of polymer-boundcarbon black in a silica gel matrix improves solvent uptake. Hydrogenbonds are formed between carbonyl groups in grafted polymer and residualsilanol groups in the silica gel network. Thus carbon blackmacromolecular imprinted silica particles are synthetized.

Since antibodies recognize target molecules by multiple weakelectrostatic, hydrophobic, and hydrogen bonding interactions betweenthe antigen binding site and the paratope of the antibody, functionalmonomers that mimic such interactions are needed. For the silica gelmatrix, tetraethyl orthosilicate (TEOS) was used as the backbone monomerand 3-Aminopropyl triethoxysilane (APS) as a functional monomer, whilecarbon black and 2-pyrrolidone also have the role of potentialfunctional monomers.

Because MIP synthesis occurs in aqueous media, water and its ioniccontent play an important role in the synthesis. Two waterconcentrations were used for each type of reaction using deionizedwater. The reactions were then repeated using 0.2M MES saline bufferinstead of deionized water. Furthermore, it is typical for Stöberprocesses to be carded in water/alcohol/ammonia mixtures. Since ethanolis not essential for the current reactions, all MIPs syntheses werecarried with the use of ethanol, and all reactions were repeated in theabsence of ethanol in solution.

Finally sodium dodecyl sulfate (SDS) was used as a structure-directingagent, Surfactants have been used for the synthesis of mesostructuredsilica materials with large porosity made of uniform mesopores. Theimportance of mesopores in MIPs particles was seen as a way ofincreasing template absorption in the particle, while at the same timeenhancing the template removal process and limiting permanent templateencapsulation by enhancing network diffusion. To test the role ofsurfactants in the synthesis, all chemical reactions were carried in thepresence of SOS and again repeated in the absence of such.

A total of 16 MIPs were synthetized, four variables were used: waterconcentration, ionic content, ethanol presence, SDS presence. Eachvariable had two test conditions, yielding 24 reactions. Human ChorionicGonadotropin (hCG) was used as the template for all syntheses. Aftertemplate removal and particle washing, agglomeration tests wereperformed with all 16 MIP's in the presence of their template, alongsidetheir respective controls using only PBS. Each MIPs were evaluated usingan in-house built device capable of simultaneously measuring up to foursamples for near infrared transmission.

Experimental Procedure 2.1 Materials

Tetraethyl orthosilicate (TEOS) and 3-aminopropyl triethoxysilane (APS)were obtained from Sigma™. Human chorionic gonadotropin (hCG) wasobtained lyophilized from Sigma™; a solution of 20 mg/nil was preparedin PBS buffer. Sodium dodecyl sulfate was in powder from sigma and a 10%wiv solution was prepared using ultrapure water. Hydrochloric acid(37%), ethanol, anhydrous acetic acid and methanol were acquired fromFisher Scientific™. Ammonium hydroxide was obtained from sigma at 30%concentration. Polymer grafted carbon black was obtained by collectingink from HP# 33 cartridges, Average sizes of this carbon black is foundto be 15 nm and grafted with 2-Pyrrolidone as per MSDS, Ultrapure waterwas obtained from a Milli-Q Millipore unit with a water quality of atleast 18.2 MΩ. 0.2M MES buffer was obtained from Fisher Scientific™ in500 ml pouches. 1×PBS 200 ml tablets were obtained from Sigma™.

2.2. Synthesis of Imprinted Silica Particles

Imprinted silica particles were prepared by the sol-gel method. Due tothe complex reaction kinetics of the silica sol-gel process, amultivariable test was performed in order to better understand the roleof key reagents in the mixture. Variables for the batch process were:water content: high or low, ionic content distilled or 0.2 MES, ethanolpresence: yes or no, SDS presence: yes or no. A total of 16 parallelreactions were performed.

Refinements to the order of mixtures and reagent quantities wereperformed. The order of reagent is paramount and if not properlyfollowed, nucleation and gelation might occur prior to the addition ofthe template; hence, no molecular imprinting will occur. Furthermore,ammonium hydroxide was used as a catalyzes and APS was limited to 172μl. In order to minimize template exposure and to increase control inreactions, two solutions were prepared. Solution one was devised topromote gelation over nucleation in solution and to introduce carbonblack and SDS if present. Since solution one can be stored withoutreagent consumption, two such batches were prepared beforehand with andwithout SOS as a variable: solution 1 a was composed of 8 ml of ink, 8ml of SDS, and 6.08 ml of ammonium hydroxide. Solution 1 b was composedof 8 ml of ink and 6.08 ml of ammonium hydroxide.

Solution two was devised to promote monomer nucleation, which occurs ina solution at or just below a pH of 4.7. Afterwards, functional monomerwas added, which neutralizes the solution pH. Since a neutral pH doesnot favor nucleation, TEOS is allowed to hydrolyze prior to functionalmonomer addition, as the basicity and quantity of the monomerneutralizes the reaction's pH; TEOS hydrolysis is determined by thesolution's return to room temperature. Once the solution achieves aneutral pH, the solution matches physiological conditions of thetemplate. For our particular batch, pH was left as is on all solutionsand HCG template was added.

Since all 16 reactions were synthesized in parallel of each other,individual solutions were prepared. In order to simplify the batchprocess, solutions 2 were grouped as follows:

TABLE 1 Group 1 Solution Water Quantity Ionic content Alcohol SDS 2A LowDeionized YES YES* 2B Low Deionized YES NO^(†) 2C Low Deionized NO YES*2D Low Deionized NO NO^(†)

TABLE 2 Group 2 Solution Water Quantity Ionic content Alcohol SDS 2E Low0.2M MES YES YES* 2F Low 0.2M MES YES NO^(†) 2G Low 0.2M MES NO YES* 2HLow 0.2M MES NO NO^(†)

TABLE 3 Group 3 Solution Water Quantity Ionic content Alcohol SDS 2IHigh Deionized YES YES* 2J High Deionized YES NO^(†) 2K High DeionizedNO YES* 2L High Deionized NO NO^(†)

TABLE 4 Group 4 Solution Water Quantity Ionic content Alcohol SDS 2MHigh 0.2M MES YES YES* 2N High 0.2M MES YES NO^(†) 2O High 0.2M MES NOYES* 2P High 0.2M MES NO NO^(†)

Synthesis of Group 1

Solutions 2A-2D have two common variables, despite of this, eachsolution was prepared individually. Briefly, these solutions wereprepared using 1.5 ml of deionized water each. For solutions 2A and 2B,820 μl of ethanol was added, respectively. Then 6 μl of HCl was added toall solutions. All solutions were gently agitated by hand; reactionswere then allowed to return to room temperature before any furtherreagent addition. Afterwards, 35 μl of APS was added, followed by 25 μlof HCl. It is at this stage where pH should be neutral as determined byprior experimentation.

Afterwards, 65 μl of hCG template was added. A short amount of time isallowed for template adsorption at nucleating sites of the silica sol;solutions are gently agitated by hand during this process. Finally, 135μl of APS was added followed by the appropriate solution one; forsolutions 2A and 2C, solution 1a was used; and for solutions 2B and 2D,solution 1b was used. All solutions were topped off by adding of 40 mlof deionized water.

Synthesis of Group 2

Solutions 2E-2H were synthesized as 2A-2C with the exception of using0.2 M MES rather than deionized water.

Synthesis of Group 3

Solutions 2I-2L prepared using 2.4 ml of deionized water each. Forsolutions 2I and 2J, 820 μl of ethanol was added followed by 6 μl ofHCl. Ail solutions were gently agitated by hand; reactions were thenallowed to return to room temperature before any further reagentaddition. Afterwards, 56 μl of APS was added, followed by 25 μl of HCl.Afterwards, 100 μl of hCG template was added. A short amount of time isallowed for template adsorption at nucleating sites of the silica sol;solutions are gently agitated by hand during this process. Finally, 115μl of APS was added followed by the appropriate solution one; forsolutions 2I and 2K solution 1a and solutions 2J and 2L solution 1b wasused.

Synthesis of Group 4

Solutions 2M-2P were prepared as 2I-2L with the exception of using 0.2 MMES buffer instead of deionized water.

2.6. Template Removal

To remove unreacted monomers, resulting molecular imprinted particleswere centrifuged at 4000 RPM for 10 minutes, rinsed with 40 ml ofdeionized water in triplicate. In previous papers, imprinted particleswere allowed to dry and grinded against a stainless steel mortar untillarge agglomerates were no longer observed. This was not done becausethe carbon black limits the aggregate size and prevents largeagglomerations and most of the imprints are located at the surface ofthe particles. In short, particle grinding is not necessary forparticles that are easily suspended in water and remain colloidal forlonger than 5 minutes.

After triplicate rinsing, particles were washed in 40 ml of elutionsolution consisting of 50% v/v mixture of glacial acetic acid andmethanol at room temperature under sonication for 10 minutes. Particleswere then centrifuged at 4000 RPM for 10 minutes, 2 ml of supernatantwas collected per solution. Particles were then rinsed in triplicatewith 40 ml of deionized water. Absorbance of the elution supernatant wasmeasured by uv-vis spectrometry at 280 nm. If absorbance was measured inthe supernatant, this indicated unbinding of the template. If absorbancevalues were larger than 0.040, solutions were washed again in 40 ml ofelution solution and rinsed in triplicate. Finally, particles werecentrifuged at 4000 RPM for 10 minutes and suspended in a 1×PBS solutionand stored at room temperature until needed. Particle concentrationswere found to be at 58±18 mg/ml.

Synthesis of Sol-Gel Affinity Columns 2.7. Materials

Tetraethyl orthosilicate (TEOS) and 3-Aminopropyl triethoxysilane (APS)were obtained from Sigma and used as is. Hydrochloric acid (37%),ethanol, and methanol were acquired from Fisher Scientific®. Anhydrousacetic acid was also from Fisher Scientific. Polymer grafted carbonblack was obtained by collecting ink from HP 33 cartridges. According toliterature, carbon black are 15 nm carbon particles [7] grafted with2-Pyrrolidone as per HP MSDS. Ultrapure water was obtained from aMilli-Q Millipore unit with a water quality of at least 18.2 MΩ. 0.1MMES buffer was obtained as powder pouches from Fisher Scientific®. 1×PBS200 ml tablets were obtained from Sigma®. Elution buffer was made with0.15 M NaCl and 0.5% acetic acid. West Nile antibodies (AWN) whereobtained from mouse ascitic fluid. Antibodies where purified by antibodyaffinity gel goat affinity purified antibody to mouse IgG column from MPbio #55581. Resulting antibody is then desalted with GE Hitrap 5 mldesalting column. Biomate 3 UV-Vis spectrophotometer was used forprotein determination with BCA and micro BCA kits from FisherScientific®. Tricorn 10/50 column #28-4064-14 was used for HPLC testing.Pierce Centrifuge columns #89896 where used as chromatography columnsfor MIPs synthesized. HPLC UNIT

2.8. Synthesis of Imprinted Silica Particles

Imprinted silica particles were prepared by the sol-gel method. In orderto maximize particle retention, a two-step synthesis protocol wasdevised. The main protocol focuses on molecular imprinting whichproduces a broad range of molecular imprinted particles in the nanometerscale. In order to contain these, a silica gel is produced where theparticles are suspended and trapped in a silica gel matrix resulting infewer particle losses during washing and regeneration protocols. Theorder of reagents is of utmost importance and if not properly followed,nucleation and gelation might occur prior to the addition of thetemplate, thus reducing or inhibiting molecular imprinting. It is notedthat all disclosed reactions are scalable.

For the synthesis of MIPs: 365 μl of 2× MES was added to a 1.5 mlconical centrifuge tube, followed by 146 μl of TEOS, then 11.8 μl ofHCl, afterwards 45 μl of APS and 11.3 μl of cAPS applied by vigorouslypipetting, then 222 μl of AWN with a concentration of 100 μg/ml,followed by 62.5 μl of ink, solution was mixed by vigorously pipettingthe solution for at least 30 seconds. Afterwards, solution is leftovernight.

For MIP particle encapsulation: 487 μl of TEOS is added to a 5 ml glasstube, MIP particles are resuspended in solution and the entire contentsare added to the glass tube, 97.3 μl of APS and 24.3 μl of cAPS are thenadded and mixed by pipetting vigorously, followed by 208 μl of ink, andfinally 684 μl of ethanol are added. After an hour, the solution becomesa gel which is then transferred to a HPLC column or a centrifuge columndepending on test.

Particles were loaded to an HPLC column for non-specificity and proteinretention testing. This was accomplished by transferring gel duringcuring time before the gelation point or after gelation by resuspendinggel in 1 ml of 2×PBS and pipetting the gel to the column. A total of 3MIP columns and 3 non-imprinted (NIP) columns were created. Columnpacking was achieved by monitoring column backpressure to no more than150 psi. Depending on batch, flow rates varied from 0.6 ml/min to 1ml/min. If backpressure was exceeded for extended periods, columnclogged and repacking was necessary.

After column packing, MIP columns were equilibrated with 2×PBS untilabsorbance at 280 nm stabilized. Afterwards, 20 μl of AWN serum wasinjected through the sample injector. After column equilibration, bufferwas changed to elution buffer in order to elute retained antibody fromthe column. In case of protein detection, sample was collectedimmediately from the detector's output. For NIP columns, no protein wasdetected during protein elution; therefore, samples were not collected.All samples from MIP columns collected were processed with a desaltingcolumn for buffer exchange to 1×PBS buffer. Mouse ascitic fluid,purified antibody from the affinity column, and collected samples werethen tested with a western blot.

For antibody activity, particles were loaded to centrifuge columns bytransferring MIP gel after suspending in 1 ml 2×PBS. A total of X MIPcolumns and Y NIP columns were fabricated. Each column was loaded with800 μl of gel and 4 ml of elution buffer. Columns were then centrifugedat 180 g's for 5 minutes. Compacted gel was resuspended with remainingsolution with a vortex. Columns were centrifuged and steps were repeateduntil columns were free of solution. A total of 12 ml of elution bufferwas used; no protein was detected in final elution with micro BCA kit.Columns were then stabilized with 6 ml of 2×PBS in the same manner asbefore.

For column testing, 60 μl of AWN serum was added to columns along with800 μl of 2×PBS. Gel was resuspended with a vortex and columns wereincubated for 3 hours. After incubation, columns were centrifuged for 5minutes at 180 g's, eluent was discarded. Column was then washed with Xml of 2×PBS to remove nonspecific bound molecules. To elute antibodyfrom the column, 800 μl of elution buffer was added. Gel was resupended,centrifuged, and sample was collected. Elution step was repeated andboth elution samples were added. Afterwards, samples were then changedto 1×PBS buffer solution with a desalting column. Up to 500 μl of thesamples were tested for protein with BCA and micro BOA kits depending onprotein concentration. Samples were also tested with ELISA for antibodyactivity and concentration.

3. RESULTS

After synthesis of all MIP, data was obtained as explained below.

3.1. Imprinted Particles Color Intensity

After template removal, black color intensity was observed differentfrom groups 1 and 2 when compared against groups 3 and 4. In order toquantify color intensity, images were recorded and black saturationpercentage measured using Adobe™ Photoshop™ CS5.1. Results aresummarized in Table 5.

TABLE 5 Black Color Intensity Group 1 Group 2 Group 3 Group 4 ParticlesColor % Color % Color % Color % A|E|I|M 95 95 80 90 B|F|J|N 95 95 80 85C|G|K|O 95 95 90 85 D|H|L|P 95 95 80 85

3.2. Reagent Retention and Template Removal

During template removal process, batch groups 1 and 2 where particularlydifficult to process. These set of particles better retained reagentsand SDS as well. Because of this, group 1 and 2 particles required anextra washing procedure when compared to groups 3 and 4. These samplesalso showed darker color intensity. Thus, we conclude that higher carbonblack concentrations enhances solvent uptake, but at the same time thissuppresses washing effectiveness probably by increasing networkcrosslinking.

In addition of being required for molecular imprinting, excessiveconcentrations of polymer grafted carbon black will slow the gelationprocess which is necessary for molecular imprinting to occur. Thus, weconclude that there is a best concentration range of carbon blackapproximately between 0.5% and 1.5%. This is because carbon black mustrender enough material to assist in molecular imprinting while at thesame time the effect of reagent uptake and the suppression of thegelation process must be limited.

3.4. Rate of Precipitation

In order to quantify precipitation rates, a device was built with 4sensors capable of reading infrared transmission changes per time in astandard disposable cuvette. The device is capable of collecting changesfrom full dispersion up to almost full precipitation with a resolutionof 1024 bits. The device was programed to collect values of all sensorssimultaneously every 2 seconds and values where recorded and transferredto a spread sheet for processing and evaluation.

For rate of precipitation tests, 2 mg of all batch particles wherecollected in solutions individually. Then 400 μg of hCG was added toeach. Finally the solutions where suspended in a total volume of 3 ml in1×PBS solution. The same process was repeated, but with the absence ofhCG as a control value of rate of precipitation per solution. Tests wereperformed in pairs of solutions with their respective template andcontrol per particle type.

FIG. 3 illustrates an exemplary graphical illustration 300 of the nearIR transmission through a cuvette in the presence of dispersed beads foreach pair as function of time, in accordance with the disclosedembodiments. As can be seen from the figure, all solutions block nearlyall transmission initially, but become more transparent as the particlessettle due to gravity and agglomeration. The rate of precipitation isdetermined by the slope of the curves. As each batch will have aslightly different precipitation rate, the ratio of the rates when hCGwas added to the rates in solvent alone were calculated. A successfulimprint was determined if a ratio equal or greater than 2 was observed,meaning that the rate of precipitation for the template containingsolution must be at least twice as fast as the rate of precipitation ofits own control.

Table 6 summarizes the precipitation rates for 16 samples.

TABLE 6 Ratio of Template Over Control Decay Slopes. Group 1 Group 2Group 3 Group 4 Particles Ratio Ratio Ratio Ratio A|E|I|M 1 2 4.15 5.08B|F|J|N 1 1 4.22 1 C|G|K|O 1 1 1 1 D|H|L|P 1 1 2.83 N/A N/A: Estimatedvalues from prior observations. Particles were not available for testingwith device.

For group 1, there was no successful imprinting in any of the particlesA to D. In group 2, only particles 2E were successful in rebinding theirtemplate, but had the smallest slope ratio of all other successfulimprints. In group 3, particles 21, 21 and 2L would rebind their target,whereas 3L had the least slope ratio from the group and second smallestfrom the batch. In group 4, particles 2M and 2P rebound to theirtemplate and particles 2M showed the greatest measured slope ratio fromall other particles in the batch. Even though particles 2P were notmeasured by the device as the particles were exhausted prior to thedevice being ready for use, prior observational experiments would pointto a very high ratio of precipitation slopes,

3.4. Discussion

From FIG. 3, it can be seen that groups 3 and 4 precipitated faster thanparticles in group 2. These results are in agreement with the previousreasoning that an optimal carbon black ratio must be found in order toachieve good rebinding and good particle dispersion. If controlparticles remain for an indefinite amount of time under dispersion, thenit will be difficult to achieve precipitation of template loadedparticles since dispersion is widely favored by the 2-Pyrrolidone.

Particles produced in the presence of SDS and ethanol yielded a total of3 out of 4 successful imprints, whereas particles produced in theabsence of SDS and ethanol produced a total of 2 out 4 successfulimprints. For particles produced in the presence of ethanol and theabsence of SDS, only particle J imprinted successfully. No particleswhere successfully imprinted for those in the presence of SDS and theabsence of ethanol.

Based on these observations, it is clear that SDS is not a requiredreagent as thought of before, but it does play a role in the reactionincreasing the chance of a successful imprint. It is then believed thatan optimal SDS quantity must be used for templates that are able totolerate low doses of SOS, whereas if a delicate template is used SDScould be avoided. The role of ethanol is yet unclear, it seems to bemore important when SOS is also used, and potentially it acts as adispersant of SDS.

Water content was found to be particularly important for the materialyield of the synthesis, where group 1 and 2 had the least material andgroup 3 and 4 produced the most material. Furthermore, ionic contentrole was found more difficult to interpret since deionized waterproduced the same amount of successful imprints as those produced in 0.2MES. Despite of this, 0.2 MES is believed to be the best alternative as2 of its 3 successful imprints produced the largest slope ratios fromthe batch. Furthermore, all particles produced under 0.2 MES producedstraight slopes whereas particles produced with deionized water hadsteps in each of the template containing solutions with successfulimprinted particles.

Finally, a new imprinting method is proposed for this particular sol-gelprocess, where molecular imprinting occurs at the gelation process andnot in the nucleation of the monomers; hence, produced MIPs are not bulkparticles but rather a new type of MIP were template imprinting occursat the aggregation sites between primary particles. This imprintingmethod could be thought of as a new type of surface imprinting.

4. CONCLUSIONS

The effect of water, quantity, ionic content, SOS, and ethanol in thereaction environment to synthesize of molecularly imprinted particleswas investigated. The ability of the MIPs to rebind their target wasevaluated by measuring sedimentation rates. While no clear rules can bederived yet, the amount of water is critical, the amount of carbon blackshould be below 1.5%, both SDS and alcohol should either be presenttogether or absent, and it is preferable to use MEP over deionizedwater. Resulting MIPs are surface imprinted and can rebind their target.Under the best reaction conditions tested to date, the MIPs willprecipitate by a factor of 5 times faster in the presence of theirtemplate that without it. When sealed from the environment, the MIPs arestable for months, which may make them very attractive for use assensors or vaccines in low resource or defense applications.

MIPs Affinity 10⁻³-10⁻¹⁰ M Application Organic or aqueous media Capacity~0.1-10 μmol/g Cost $10's for g quantities Production 2-3 daysReusability 100's of times Stability Wide temperature and pH rangeStorage time Stable over period of years

FIG. 4A illustrates an exemplary pictorial illustration 400 of SEMimages demonstrating surface morphology, specifically black anti-WNparticles at 1200×, showing macroparticle shape, in accordance with thedisclosed embodiments. FIG. 4B illustrates an exemplary pictorialillustration 450 of SEM images demonstrating surface morphology,specifically black anti-WN particles at 22,000× illustrating aggregatemorphology of macroparticles, in accordance with the disclosedembodiments.

FIG. 5 illustrates an exemplary pictorial illustration 500 ofagglomeration test of UV red, carbon black, and non-colored silicaparticles, in accordance with the disclosed embodiments. A, D, G samplesare UV red, carbon black, and non-colored silica particles in thepresence of anti-WN template respectively. B, E, H samples are UV red,carbon black, and non-colored silica particles in the presence of HCG asa false positive control. C, F, I samples are UV red, carbon black, andnon-colored silica particles in PBS as standard control.

FIG. 6 illustrates an exemplary pictorial illustration 600 of conicaltubes containing imprinted silica particles that were re-suspended afterovernight precipitation, in accordance with the disclosed embodiments.In FIG. 6: (A) indicates anti-West Nile imprinted silica particles inpresence of mouse anti-WN antibody containing serum; (B) indicatesanti-WN beads in PBS; (C) indicates WNP in presence of mouse anti-Dengueantibody containing serum; (D) indicates hCG imprinted silica particlesin presence of mouse anti-WN antibody containing serum; and (E)indicates hCG imprinted silica particles in PBS.

Based on the foregoing, it can be appreciated that a number of differentembodiments, preferred and alternative are disclosed herein. Forexample, in one embodiment, a method of imprinting silica beads can beimplemented. The method can include adding polymerized silica, aprecursor, and carbon black in a presence of a molecular template;polymerizing the polymerized silica, the precursor, and the carbon blackaround the molecular template and forming a polymerized matrix bead; andwashing the molecular template out of the polymerized matrix bead,wherein an imprint of the precursor remains in the polymerized matrixbead as an imprinted matrix space.

In some embodiments, the silica comprises macromolecular imprintedsilica particles. In other embodiments, the precursor comprises at leastone of an antibody, a virus, a protein, a hormone, an antigen, anenzyme, a molecule, a molecule with a molecular weight less than orequal to 1500 Da, and a molecule with a molecular weight greater than1500 Da. In other embodiments, the molecular template leaves achemically and sterically complementary void or imprint in thepolymerized matrix bead, wherein the void rebinds the moleculartemplate. In yet another embodiment, a step can be implemented forutilizing a silica gel matrix and tetraethyl orthosilicate as a backbonemonomer and 3-aminopropy/triethoxysilane as a functional monomer,wherein the imprinted silica particles are prepared utilizing sol-gelaffinity columns, further comprising utilizing sodium dodecyl sulfate(SDS) as a structure-directing agent by forming mesopores that increasetemplate absorption, enhance molecular template removal, and limitpermanent template encapsulation by enhancing network diffusion.

In other embodiments, a step can be implemented for imprinting theprecursor in the polymerized matrix bead and forming a colored polymerbead of a preferred diameter range measuring from 1 micron to 8 microns.In another embodiment, a step can be implemented for synthesizingimprinted materials in a presence of polymer-grafted carbon black,wherein the imprinted materials rebind high molecular weight templates.In yet another embodiment, a step can be implemented for injecting theimprinted polymerized matrix bead into a living specimen and the livingspecimen selectively producing antibodies that fill the imprinted matrixspace, thus creating a vaccine.

In yet another embodiment, an imprinted silica bead is disclosed. Theimprinted silica bead can include polymerized silica, a precursor, andcarbon black in a presence of a molecular template, wherein an imprintof the precursor remains in the polymerized matrix bead as an imprintedmatrix space. In an embodiment, the silica comprises macromolecularimprinted silica particles. In yet another embodiment, the precursorcomprises at least one of an antibody, a virus, a protein, a hormone, anantigen, an enzyme, a molecule, a molecule with a molecular weight lessthan or equal to 1500 Da, and a molecule with a molecular weight greaterthan 1500 Da. In other embodiments, the molecular template comprises achemically and sterically complementary void or Imprint in thepolymerized matrix bead, wherein the void is capable of rebinding themolecular template. In an embodiment, the precursor is imprinted in thepolymerized matrix bead. In yet other embodiments, the colored polymerbead has a preferred diameter range measuring from 1 micron to 8microns.

In another embodiment, an imprinted silica microbead is disclosed. Theimprinted silica microbead can include polymerized silica; a moleculewith a high molecular weight imprinted in a location of the polymerizedsilica; and an artificial receptor at the location of the imprintedmolecule, wherein the artificial receptor selectively binds molecules.In an embodiment, the silica comprises macromolecular imprinted silicaparticles. In yet other embodiments, the precursor comprises at leastone of an antibody, a virus, a protein, a hormone, an antigen, anenzyme, a molecule, a molecule with a molecular weight less than orequal to 1500 Da, and a molecule with a molecular weight greater than1500 Da. In other embodiments, the molecular template comprises achemically and sterically complementary void or imprint in thepolymerized matrix bead, wherein the void is capable of rebinding themolecular template. In yet another embodiment, the precursor isimprinted in the polymerized matrix bead. In an embodiment, the coloredpolymer bead has a preferred diameter range measuring from 1 micron to 8microns.

It will be appreciated that variations of the above-disclosed and otherfeatures and functions, or alternatives thereof, may be desirablycombined into many other different systems or applications. Furthermore,various presently unforeseen or unanticipated alternatives,modifications, variations or improvements therein may be subsequentlymade by those skilled in the art which are also intended to beencompassed by the following claims.

What is claimed is:
 1. A method of imprinting silica beads, comprising:adding polymerized silica, a precursor, and carbon black in a presenceof a molecular template; polymerizing said polymerized silica, saidprecursor, and said carbon black around said molecular template andforming a polymerized matrix bead; and washing said molecular templateout of said polymerized matrix bead, wherein an imprint of saidprecursor remains in said polymerized matrix bead as an imprinted matrixspace.
 2. The method of claim 1 wherein said silica comprisesmacromolecular imprinted silica particles.
 3. The method of claim 1wherein said precursor comprises at least one of an antibody, a virus, aprotein, a hormone, an antigen, an enzyme, a molecule, a molecule with amolecular weight less than or equal to 1500 Da, and a molecule with amolecular weight greater than 1500 Da.
 4. The method of claim 1 whereinsaid molecular template leaves a chemically and sterically complementaryvoid or imprint in said polymerized matrix bead, wherein said voidrebinds said molecular template.
 5. The method of claim 1 furthercomprising utilizing a silica gel matrix and tetraethyl orthosilicate asa backbone monomer and 3-aminopropy/triethoxysilane as a functionalmonomer, wherein said imprinted silica particles are prepared utilizingsol-gel affinity columns, further comprising utilizing sodium dodecylsulfate (SDS) as a structure-directing agent by forming mesopores thatincrease template absorption, enhance molecular template removal, andlimit permanent template encapsulation by enhancing network diffusion.6. The method of claim 1 further comprising: imprinting said precursorin said polymerized matrix bead; and forming a colored polymer bead of apreferred diameter range measuring from 1 micron to 8 microns.
 7. Themethod of claim 1 further comprising synthesizing imprinted materials ina presence of polymer-grafted carbon black, wherein said imprintedmaterials rebind high molecular weight templates.
 8. The method of claim1 further comprising injecting said imprinted polymnerized matrix beadinto a living specimen and said living specimen selectively producingantibodies that fill said imprinted matrix space, thus creating avaccine.
 9. An imprinted silica bead, comprising: polymerized silica, aprecursor, and carbon black in a presence of a molecular template,wherein an imprint of said precursor remains in said polymerized matrixbead as an imprinted matrix space.
 10. The imprinted silica bead ofclaim 9 wherein said silica comprises macromolecular imprinted silicaparticles.
 11. The imprinted silica bead of claim 9 wherein saidprecursor comprises at least one of an antibody, a virus, a protein, ahormone, an antigen, an enzyme, a molecule, a molecule with a molecularweight less than or equal to 1500 Da, and a molecule with a molecularweight greater than 1500 Da.
 12. The imprinted silica bead of claim 9wherein said molecular template comprises a chemically and stericallycomplementary void or imprint in said polymnerized matrix bead, whereinsaid void is capable of rebinding said molecular template.
 13. Theimprinted silica bead of claim 9 wherein said precursor is imprinted insaid polymerized matrix bead,
 14. The imprinted silica bead of claim 9wherein said colored polymer bead has a preferred diameter rangemeasuring from 1 micron to 8 microns.
 15. An imprinted silica microbead,comprising: polymerized silica; a molecule with a high molecular weightimprinted in a location of said polymerized silica; and an artificialreceptor at said location of said imprinted molecule, wherein saidartificial receptor selectively binds molecules.
 16. The imprintedsilica bead of claim 15 wherein said silica comprises macromolecularimprinted silica particles.
 17. The imprinted silica bead of claim 15wherein said precursor comprises at least one of an antibody, a virus, aprotein, a hormone, an antigen, an enzyme, a molecule, a molecule with amolecular weight less than or equal to 1500 Da, and a molecule with amolecular weight greater than 1500 Da.
 18. The imprinted silica bead ofclaim 15 wherein said molecular template comprises a chemically andsterically complementary void or imprint in said polymerized matrixbead, wherein said void is capable of rebinding said molecular template.19. The imprinted silica bead of claim 15 wherein said precursor isimprinted in said polymerized matrix bead.
 20. The imprinted silica beadof claim 15 wherein said colored polymer bead has a preferred diameterrange measuring from 1 micron to 8 microns.